甘藷塊根L型澱粉磷解酶不需醣引子合成醣前體之活性催化機制

The Catalytic Mechanism of Primer-independent Glucan Precursor-Synthesizing Activity of L-Form Starch Phosphorylase from Sweet Potato Roots

林之儀(Chih-yi Lin)

指導教授 ： 莊榮輝

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.03239

摘要

L型澱粉磷解酶 (L-SP) 為存在於高等植物造粉體之澱粉代謝酵素，同時具有磷解澱粉以及合成澱粉雙方向之活性。在in vitro可以利用基質Glc-1-P於不含醣引子 (primer-independent, PI) 或者是含醣引子 (primer-dependent) 之情況下進行直鏈醣合成。使用HPAEC (high performance anion exchange chromatograph) 分析發現，L-SP的PI反應之速率決定步驟為將兩個單醣合成雙醣，而當Glc-1-P合成雙醣或寡醣後，反應即進入快速延長時期，此時磷酸釋放以及直鏈醣生成速率皆大幅上升。此外，本論文發現在L-SP上與Glc-1-P結合的兩個區域，其中之一對於基質辨認之專一性較高，僅能與Glc-1-P結合 (在活性區上)；另一個結合區可能在L-SP之特殊插入序列L78上，可接受不同的基質，故可與Glc-1-P以外之六碳醣磷酸結合。利用L-SP之同功酶H-SP進行不含醣引子之合成反應，發現其不具PI之活性。由上述結果推論，L-SP之催化機制應該是由PLP (pyridoxal phosphate) 結合區上之Glc-1-P先裂解生成碳陽離子後，再去攻擊與L78結合之第二個Glc-1-P的非還原端，形成

關鍵字

澱粉磷解酶澱粉代謝 ； 不含醣引子合成 ； 葡萄糖-1-磷酸 ； 高效能陰離子交換層析法 ； 磷酸吡哆醛

並列摘要

L-Form starch phosphorylase (L-SP) is a plastidial alpha-glucan phosphorylase in higher plants which catalyzes reversible reactions of starch synthesis and degradation. In vitro, the enzyme might synthesize linear glucan in the absence of a primer (primer-independent activity, PI activity). By analyzing glucan products with HPAEC (high performance anion exchange chromatograph), we found that the rate-determining step in the PI activity was the formation of disaccharide from two molecules of Glc-1-P. The reaction went into rapid elongation phase after disaccharides or oligosaccharides were produced. At this stage, the rate of the phosphate releasing and linear glucan synthesizing increased exponentially. In each cycle of catalytic reaction, one Glc-1-P bound to the active site on L-SP with high affinity which was then reacted with the other molecule of Glc-1-P on L78, the insertion region on L-SP. The basic amino acids on L78 could bind with Glc-1-P or short glucan to serve as the second substrate binding site. It was postulated that the catalytic mechanism for L-SP might follow these steps: The Glc-1-P anchored to PLP (pyridoxal phosphate) in binding site and was cleaved into carbocation, then attacked the nonreducing end of the second Glc-1-P or glucan on L78, forming

並列關鍵字

starch phosphorylase ； starch synthesis ； primer-independent ； PI ； Glc-1-P ； HPAEC-PAD ； PLP

參考文獻

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